Plasma levels of the calcium regulating hormone 1,25-(OH)2Vitamin D3 are strictly regulated by both calcium and phosphate and the effects of these regulatory factors are probably mediated by changes in the rate of renal synthesis of the hormone. Several physiological studies in animals as well as in vitro studies in cultured avian kidney cells have shown that regulation of the 1Alpha-hydroxylase by calcium is probably mediated by PTH and cAMP, unlike regulation by phosphate which requires the presence of an intact pituitary and growth hormone. Despite all of this information, very little is known about how the activity of the enzyme is stimulated at the molecular level. Studies in the avian system have shown that the enzyme complex is composed of a mixed function-oxidase which contains cytochrome P-450 as its terminal component. It also contains a ferredoxin and ferredoxin reductase which pass electrons from NADPH to the cytochrome P-450. However, there is controversy about whether a similar system exists in mammalian kidney, and the mammalian enzyme has been less extensively studied, primarily due to technical difficulties in measuring the low rates of in vitro 1,25-(OH)2D3 production, especially in kidney preparations from vitamin D-replete animals. Using newly developed more sensitive techniques to measure in vitro 1,25-(OH)2D3 production, the principal objective of the present project will be to determine whether there is a requirement for a ferredoxin and ferredoxin reductase in a solubilized, reconstituted 1Alpha-hydroxylase system containing a mammalian cytochrome P-450 which has been partially purified by p-chloroamphetamine-sepharose chromatography. Further purification of the cytochrome P-450 will be accomplished by ion exchange chromatography in detergent-containing buffer and substrate affinity chromatography using the 3Beta-hemisuccinate derivative of 25-OH-D linked to sepharose 4Beta. Since 25-OH-D can be converted to a number of products in vitro, identification of the products generated in reconstitution assays with the purified cytochrome P-450-1Alpha will be carried out using mass spectroscopy. Longterm objectives of this project will be to generate specific antibodies to the purified P-450-1Alpha which will permit quantitation of P-450-1Alpha content in kidney tissue from animals in which the activity of the 1Alpha-hydroxylase has been stimulated. These studies should also make it possible to determine whether there is a single form or multiple forms of P-450-1Alpha in the kidney during various forms of physiological stress.